title: Novel single-step multiplex real-time polymerase chain reaction assay for simultaneous quantification of hepatitis virus A, B, C, and E in serum.
abstract: OBJECTIVE
  Viral hepatitis needs an earliest diagnosis for its proper and timely treatment. Although serodiagnosis of viral hepatitis is in regular practice, however, it has certain limitations and points to alternate procedures of diagnosis. Present study was designed to develop a single-step multiplex real-time polymerase chain reaction (PCR) assay for detection of hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV) and hepatitis E virus (HEV) related nucleic acids in sera from infected patients.
  
  
  METHODS
  The PCR was standardized to detect HAV, HBV, HCV and HEV in serum using variables including annealing temperature, extension temperature, MgCl2 , and primer concentrations. The conserved regions of all viral genomes were used as targets for amplification.
  
  
  RESULTS
  This novel assay was found to be a fast, sensitive, specific, and reproducible system for detection of HAV, HBV, HCV, and HEV in serum. The detection limit for different viral genomes at 100% level was found to be 280 copies/mL for HAV, 290 copies/mL for HBV, 30 copies/mL for HCV, and 300 copies/mL for HEV in a single-tube assay system.
  
  
  CONCLUSIONS
  Present multiplex real-time PCR is the first report on single-step nucleic acid detection of HAV, HBV, HCV, and HEV in sera samples. It is an alternate diagnostic assay for common use in laboratories analyzing viral hepatitis cases.
authors.affiliation: Clinical Biochemistry Division, Department of Laboratory Medicine, All India Institute of Medical Sciences, New Delhi, India.
authors.forename: Mohammad
authors.initials: M
authors.lastname: Irshad
authors.forename: Mohammad Ahmad
authors.initials: MA
authors.lastname: Ansari
authors.forename: Khushboo
authors.initials: K
authors.lastname: Irshad
authors.forename: Raghavendra
authors.initials: R
authors.lastname: Lingaiah
mesh_terms: D000328:Adult; D000914:Antibodies, Viral / Q000097:blood; D015415:Biomarkers / Q000097:blood; D004279:DNA, Viral / Q000097:blood; D016174:Hepacivirus / Q000302:isolation & purification; D030041:Hepatitis A virus / Q000302:isolation & purification; D006514:Hepatitis B Surface Antigens / Q000097:blood; D006515:Hepatitis B virus / Q000302:isolation & purification; D016752:Hepatitis E virus / Q000302:isolation & purification; D006518:Hepatitis Viruses / Q000302:isolation & purification*; D006525:Hepatitis, Viral, Human / Q000175:diagnosis*; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D000914:Antibodies, Viral; D015415:Biomarkers; D004279:DNA, Viral; D006514:Hepatitis B Surface Antigens
keywords: diagnosis; hepatitis; hepatitis viruses; multiplex polymerase chain reaction; real-time polymerase chain reaction